In each of 29 patients with infectious mononucleosis (IM) the development of antibodies against a herpes-type virus (EB virus) has been demonstrated by an indirect immunofluorescence test with a virus bearing cell line (EB-3) derived from a Burkitt lymphoma. These antibodies, absent in pre-illness serum specimens, usually appeared early in the disease, rose to peak levels within a few weeks, and remained at relatively high levels during convalescence. They are clearly distinctive from heterophile antibodies and, unlike the latter, they persist for years, probably for life. Tests on sera from 50 randomly selected college freshmen revealed EB virus antibodies in 12, two of whom had positive histories of IM. Of 38 without demonstrable antibodies none had had IM, but the illness developed in three in the next two years. These and other observations strongly indicate that EB virus, or a closely related one, is the etiologic agent of IM.